Pancreatic cancer is a highly aggressive malignancy that is resistant to most chemotherapeutic agents. Nucleoside analogs are currently used as the treatment of choice despite their suboptimal benefits. Our long-term goal is to improve the chemotherapeutic management of pancreatic cancer. The overall objective of this application is to elucidate the role of miR-663 and -4787 in pancreatic cancer chemoresistance. We hypothesize that the restoration of miR-663 and -4787, miRs epigenetically silenced in pancreatic cancer, will target a TGF-?et-7 axis to potentiate the nucleoside analog chemotherapeutic efficacy. This research has important translational significance because it will enable further clinical studies to improve pancreatic cancer patient sensitivity to therapy. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims. Specific aim 1 will determine the epigenetic regulation of miR-663 and -4787 in pancreatic cancer. The working hypothesis states that miR-663a and -4787-5p are silenced in pancreatic cancer consequent to histone methylation-dependent chromatin changes. In this aim, the tumor chromatin landscape will be investigated by a) studying the epigenetic silencing marks in the promoter regions of miR-663 and -4787, b) identifying epigenetic protein complexes regulating miR-663 and -4787, and c) evaluating the cancer control consequences after manipulation of epigenetic marks and histone writers by knockdown and pharmacological approaches. Specific aim 2 will elucidate the mechanism of miR-663 and -4787-induced chemosensitization. The working hypothesis is that the chemosensitization activities of miR-663 and -4787 are mediated by a reduction in epithelial-mesenchymal transition (EMT)-driven drug resistance via a TGF-let-7 axis. In this aim, the detailed biochemical mechanisms of miR-663 and -4787 regulation of TGF-?expression and let-7 subtypes' maturation will be investigated to understand their role in EMT-directed chemoresistance. In addition, the impact of miR-663 and -4787 in the innate and acquired nucleoside analog resistance will be determined. Specific aim 3 will evaluate the in vivo efficacy of miR-663 and -4787 in pancreatic tumor growth control. The working hypothesis is that the tumor delivery of miR-663 and -4787 will potentiate gemcitabine efficacy in mice carrying orthotopic pancreatic cancer xenografts. Overexpression and knockdown of miR-663 and -4787, novel nucleoside analog-oligonucleotide (miR) nanoparticle formulations and hydrodynamic delivery to the mouse tumors and pharmacokinetics will be evaluated. This approach offers a substantive innovation in the field of pancreatic cancer chemotherapy by directing co-delivery of a novel epigenetic chemoresistance inhibitor with the gemcitabine chemotherapeutic for optimized cancer cell destruction. The proposed research is significant because it directly addresses the overriding cause of nucleoside analog treatment failure in pancreatic cancer patients, chemoresistance, and advances an alternate epigenetic-chemotherapeutic combination approach to preclinical testing to overcome the limitations of current chemotherapy.